The role of titin in eccentric muscle contraction · The Journal of Experimental Biology...
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The Journal of Experimental Biology © 2014. Published by The Company of Biologists Ltd | The Journal of Experimental Biology (2014) 217, 2825-2833 doi:10.1242/jeb.099127 2825 ABSTRACT Muscle contraction and force regulation in skeletal muscle have been thought to occur exclusively through the relative sliding of and the interaction between the contractile filaments actin and myosin. While this two-filament sarcomere model has worked well in explaining the properties of isometrically and concentrically contracting muscle, it has failed miserably in explaining experimental observations in eccentric contractions. Here, I suggest, and provide evidence, that a third filament, titin, is involved in force regulation of sarcomeres by adjusting its stiffness in an activation-dependent (calcium) and active force-dependent manner. Upon muscle activation, titin binds calcium at specific sites, thereby increasing its stiffness, and cross-bridge attachment to actin is thought to free up binding sites for titin on actin, thereby reducing titin’s free-spring length, thus increasing its stiffness and force upon stretch of active muscle. This role of titin as a third force regulating myofilament in sarcomeres, although not fully proven, would account for many of the unexplained properties of eccentric muscle contraction, while simultaneously not affecting the properties predicted by the two- filament cross-bridge model in isometric and concentric muscle function. Here, I identify the problems of the two-filament sarcomere model and demonstrate the advantages of the three-filament model by providing evidence of titin’s contribution to active force in eccentric muscle function. KEY WORDS: Cross-bridge theory, Eccentric contraction, Force enhancement, Instability, Popping sarcomere hypothesis, Sarcomere, Titin Introduction Eccentric muscle contractions are defined as contractions in which an active muscle is stretched. Because muscles tend to shorten upon activation, eccentric contractions occur when the external forces acting on a muscle are greater than the forces produced by the muscle. Although eccentric contractions may be thought of as an anomaly, they occur during most everyday movements. For example, when descending a set of stairs, our knee extensor muscles are active to control the movement, but only to a degree that produces forces that are smaller than the forces acting on the muscle by gravity as we walk down the stairs. While isometric (constant muscle length) and concentric contractions (muscle is actively shortening) are reasonably well described and explained by current theories of muscle force production (Huxley, 1957), eccentric contractions are not. Andrew Huxley, who first formulated the cross-bridge theory of contraction (the current paradigm for the molecular mechanism underlying muscle activation and force production), realized this when forces and energy requirements of eccentric contractions predicted by his COMMENTARY Human Performance Lab, Faculty of Kinesiology, University of Calgary, Calgary, AB T2N 1N4, Canada. *Author for correspondence ([email protected]) theory vastly exceeded the experimentally observed values (Hill, 1938; Abbott and Aubert, 1951; Joumaa and Herzog, 2013). In his book ‘Reflections on Muscle’, while discussing eccentric contractions, Huxley (Huxley, 1980) stated that: ‘Elongations of active muscle is something that happens in very many movements’ and that ‘I imagine that special features have been evolved which allow this elongation to take place without damaging the muscle’ and further that ‘these special features have little relation to the processes that take place during shortening.’ He then went on to describe that eccentric muscle contractions comprise a wide and difficult field for investigation that will hold a number of surprises. Here, I will attempt to discuss some of the difficulties associated with eccentric muscle contractions, the special features that have evolved to limit damage, and explain some of the surprising features of actively lengthening muscles. Mechanisms of contraction Prior to the 1950s, it had been assumed that muscle activation and force production were associated with the shortening of the thick filaments in the centre of sarcomeres. It was thought that activation (calcium release) caused the myosin filaments that comprise the thick, A-band filaments to be transformed from a helix-like configuration into a coil-like configuration (Fig. 1), thereby causing muscle shortening and force production. Specifically, the long myosin filaments that were aligned in long strands were thought to undergo calcium-induced shortening at specific points, thereby producing muscle activation. However, Hugh Huxley hinted at the idea that thick filaments did not shorten upon activation (Huxley, 1953), and he and Andrew Huxley then published their seminal work on the sliding filament theory (Huxley and Niedergerke, 1954; Huxley and Hanson, 1954) where they argued independently that thick filaments did not shorten. Rather, they suggested that muscle contraction occurred through the relative sliding of the thick and thin filaments (Fig. 1). Three years later, Andrew Huxley then provided the first molecular and mathematical framework of how this relative sliding of the two sets of filaments was supposed to occur: the cross-bridge theory (Huxley, 1957). His theory was based on the idea that there are uniformly arranged side projections (cross-bridges) on the thick filaments that interact cyclically with specific attachment points on the thin filaments, thereby pulling the thin past the thick filaments. These interactions by the cross-bridges were driven partially by Brownian motion and partially by the hydrolysis of adenosine triphosphate (ATP), one ATP per cross-bridge cycle. The theory proposed in 1957 had two cross-bridge states, one attached and one detached. Hugh Huxley then proposed that cross- bridge action likely involved rotation (Huxley, 1969), a concept adapted by Andrew Huxley and extended to multiple attachment states (Huxley and Simmons, 1971). This multiple-state model had the advantage that it could explain force transients following quick stretches or releases. Although cross-bridge models with more than 20 states have been described in the literature, commonly cross- bridge action and muscle contraction are described by models that The role of titin in eccentric muscle contraction Walter Herzog*
Transcript of The role of titin in eccentric muscle contraction · The Journal of Experimental Biology...
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© 2014. Published by The Company of Biologists Ltd | The Journal of Experimental Biology (2014) 217, 2825-2833 doi:10.1242/jeb.099127
2825
ABSTRACTMuscle contraction and force regulation in skeletal muscle havebeen thought to occur exclusively through the relative sliding of andthe interaction between the contractile filaments actin and myosin.While this two-filament sarcomere model has worked well inexplaining the properties of isometrically and concentricallycontracting muscle, it has failed miserably in explaining experimentalobservations in eccentric contractions. Here, I suggest, and provideevidence, that a third filament, titin, is involved in force regulation ofsarcomeres by adjusting its stiffness in an activation-dependent(calcium) and active force-dependent manner. Upon muscleactivation, titin binds calcium at specific sites, thereby increasing itsstiffness, and cross-bridge attachment to actin is thought to free upbinding sites for titin on actin, thereby reducing titin’s free-springlength, thus increasing its stiffness and force upon stretch of activemuscle. This role of titin as a third force regulating myofilament insarcomeres, although not fully proven, would account for many ofthe unexplained properties of eccentric muscle contraction, whilesimultaneously not affecting the properties predicted by the two-filament cross-bridge model in isometric and concentric musclefunction. Here, I identify the problems of the two-filament sarcomeremodel and demonstrate the advantages of the three-filament modelby providing evidence of titin’s contribution to active force ineccentric muscle function.
KEY WORDS: Cross-bridge theory, Eccentric contraction, Forceenhancement, Instability, Popping sarcomere hypothesis,Sarcomere, Titin
IntroductionEccentric muscle contractions are defined as contractions in whichan active muscle is stretched. Because muscles tend to shorten uponactivation, eccentric contractions occur when the external forcesacting on a muscle are greater than the forces produced by themuscle. Although eccentric contractions may be thought of as ananomaly, they occur during most everyday movements. Forexample, when descending a set of stairs, our knee extensor musclesare active to control the movement, but only to a degree thatproduces forces that are smaller than the forces acting on the muscleby gravity as we walk down the stairs.
While isometric (constant muscle length) and concentriccontractions (muscle is actively shortening) are reasonably welldescribed and explained by current theories of muscle forceproduction (Huxley, 1957), eccentric contractions are not. AndrewHuxley, who first formulated the cross-bridge theory of contraction(the current paradigm for the molecular mechanism underlyingmuscle activation and force production), realized this when forcesand energy requirements of eccentric contractions predicted by his
COMMENTARY
Human Performance Lab, Faculty of Kinesiology, University of Calgary, Calgary,AB T2N 1N4, Canada.
*Author for correspondence ([email protected])
theory vastly exceeded the experimentally observed values (Hill,1938; Abbott and Aubert, 1951; Joumaa and Herzog, 2013). In hisbook ‘Reflections on Muscle’, while discussing eccentriccontractions, Huxley (Huxley, 1980) stated that: ‘Elongations ofactive muscle is something that happens in very many movements’and that ‘I imagine that special features have been evolved whichallow this elongation to take place without damaging the muscle’and further that ‘these special features have little relation to theprocesses that take place during shortening.’ He then went on todescribe that eccentric muscle contractions comprise a wide anddifficult field for investigation that will hold a number of surprises.Here, I will attempt to discuss some of the difficulties associatedwith eccentric muscle contractions, the special features that haveevolved to limit damage, and explain some of the surprising featuresof actively lengthening muscles.
Mechanisms of contractionPrior to the 1950s, it had been assumed that muscle activation andforce production were associated with the shortening of the thickfilaments in the centre of sarcomeres. It was thought that activation(calcium release) caused the myosin filaments that comprise thethick, A-band filaments to be transformed from a helix-likeconfiguration into a coil-like configuration (Fig. 1), therebycausing muscle shortening and force production. Specifically, thelong myosin filaments that were aligned in long strands werethought to undergo calcium-induced shortening at specific points,thereby producing muscle activation. However, Hugh Huxleyhinted at the idea that thick filaments did not shorten uponactivation (Huxley, 1953), and he and Andrew Huxley thenpublished their seminal work on the sliding filament theory(Huxley and Niedergerke, 1954; Huxley and Hanson, 1954) wherethey argued independently that thick filaments did not shorten.Rather, they suggested that muscle contraction occurred throughthe relative sliding of the thick and thin filaments (Fig. 1). Threeyears later, Andrew Huxley then provided the first molecular andmathematical framework of how this relative sliding of the twosets of filaments was supposed to occur: the cross-bridge theory(Huxley, 1957). His theory was based on the idea that there areuniformly arranged side projections (cross-bridges) on the thickfilaments that interact cyclically with specific attachment pointson the thin filaments, thereby pulling the thin past the thickfilaments. These interactions by the cross-bridges were drivenpartially by Brownian motion and partially by the hydrolysis ofadenosine triphosphate (ATP), one ATP per cross-bridge cycle.The theory proposed in 1957 had two cross-bridge states, oneattached and one detached. Hugh Huxley then proposed that cross-bridge action likely involved rotation (Huxley, 1969), a conceptadapted by Andrew Huxley and extended to multiple attachmentstates (Huxley and Simmons, 1971). This multiple-state model hadthe advantage that it could explain force transients following quickstretches or releases. Although cross-bridge models with more than20 states have been described in the literature, commonly cross-bridge action and muscle contraction are described by models that
The role of titin in eccentric muscle contractionWalter Herzog*
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have approximately five states: two detached (pre- and post-ATPhydrolysis) and three attached (ADP·P, ADP and empty nucleotidebinding site) (Rayment et al., 1993) (Fig. 2).
The cross-bridge theory has been the paradigm for explainingthe mechanisms of contraction for the past half century. It predictsmany experimentally observed phenomena extremely well,
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GlossaryActin Contractile protein in the sarcomere comprising the main component of thinfilaments.
Active forceForce in muscle produced through the interaction of the contractile filamentsactin and myosin, and powered by ATP.
A. F. Huxley Muscle physiologist (1917–2012) who formulated the first mathematicaldescription of the cross-bridge theory in 1957.
Ascending limb The ascending limb of the force–length relationship describes the region ofmuscle function in which isometric force increases with increasing musclelength.
A. V. Hill Muscle physiologist who popularized the force–velocity property of skeletalmuscle and introduced the notion of muscle instability on the descendinglimb of the force–length relationship.
Butanedione monoxime Inhibits cross-bridges from going from the weakly to the strongly boundstate in the cross-bridge cycle.
Cross-bridge Protein side piece emanating from the thick (myosin) filament that attachesto actin in a cyclic manner.
Cross-bridge theory The current paradigm of muscle contraction. It states that musclecontraction occurs through the interaction of myosin-based cross-bridgesthat cyclically attach to specific binding sites on actin, thereby producing arelative sliding of actin past myosin that is driven by the hydrolysis of ATP.
Concentric shortening A concentric muscle contraction is one in which muscle shortens duringcontraction.
Descending limb The descending limb of the force–length relationship describes the regionof muscle function in which isometric force decreases with increasingmuscle length.
Eccentric lengthening An eccentric muscle contraction is one in which muscle is stretched whileactivated.
Fascicle Segment of muscles containing fibres that is enclosed by a distinctconnective tissue, the perimysium.
Fibre An elongated, multinucleated cell.
Force–length relationship Muscle property that describes the dependence of maximal, isometric forceon the length of the muscle.
Force–velocity relationship Muscle property that describes the dependence of maximal force at optimalmuscle length on the speed of muscle contraction.
H. E. Huxley Muscle physiologist (1924–2013) who first described the sliding filamenttheory in 1953.
Ig domain Immunoglobulin domain of titin occurring at the proximal and distal ends oftitin.
Isometric Without change in length; an isometric muscle contraction is one in whichmuscle length does not change.
M-band/line Protein band in the middle of two half-sarcomeres.
Myofibril A sub-cellular organelle comprised of serially arranged sarcomeres.
Myofilament Long muscle proteins in sarcomeres, such as actin, myosin and titin.
Myosin Contractile protein in the sarcomere comprising the main component of thethick filament.
N2A region A unique sequence within titin thought to be a possible binding site for titinwith actin.
Passive force Force in muscle not requiring energy (ATP) and primarily produced by thestructural protein titin on the myofibrillar level and also by collagen-basedconnective tissue structures on the fibre, fascicle and muscle levels.
Passive force enhancement Muscle property associated with the increase in passive force followingactive muscle stretching.
PEVK region A unique sequence within titin that is rich in proline, glutamate, valine andlysine residues.
Plateau region The plateau region of the force–length relationship describes the region ofmuscle function in which isometric force remains the same, and is maximal,for small changes in length.
Residual force enhancement Muscle property that describes the increase in steady-state isometric forcefollowing active muscle stretching compared with the corresponding purelyisometric force.
Sarcomere Smallest structural unit of contraction in skeletal muscle.
Sliding filament theory Theory of contraction that supposes that muscle contraction occurs throughthe relative sliding of the two contractile proteins actin and myosin.
Thick filament Sarcomeric contractile filament consisting primarily of myosin.
Thin filament Sarcomeric contractile filament consisting primarily of actin, troponin andtropomyosin.
Titin Structural protein in sarcomeres spanning the half sarcomere from the Z-line to the M-band.
Troponin Regulatory protein in sarcomeres.
Tropomyosin Regulatory protein in sarcomeres.
‘x’ distance The distance of the cross-bridge equilibrium position to the nearest eligibleattachment site on actin in the cross-bridge theory.
Z-line/band Protein band at the end of a sarcomere.
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particularly those involving isometric and concentric contractions,but fails to predict some of the basic and generally acceptedproperties of eccentrically contracting muscles (Pollack, 1990;Woledge et al., 1985). Some of these failures will be discussed inthe next section.
Eccentric muscle contractionsActive lengthening of muscle (eccentric contraction) is associatedwith a variety of unique properties. Force is increased comparedwith isometric or concentric contractions (Hill, 1938), energyrequirement is decreased, and forces following eccentriccontractions are increased (Abbott and Aubert, 1952). Classiccross-bridge models have difficulties predicting the force andenergy efficiency of eccentric contractions accurately (Woledge etal., 1985), and they cannot explain experimentally observedhistory-dependent properties of muscles (Walcott and Herzog,2008).
Cross-bridge models can be thought of as a series of mechanicaland/or biochemical cross-bridge states that are connected bythermodynamically consistent rate functions (Howard, 2001;Walcott and Herzog, 2008). By definition, these rate functions are
exclusively based on Huxley’s so-called ‘x’ distance, the distanceof a cross-bridge’s equilibrium position to its nearest eligibleattachment site on actin (Fig. 2A). The cross-bridge’s equilibriumposition is defined as the location at which the cross-bridge’selastic element has zero force (it is neither stretched norcompressed), while its nearest attachment site is defined as theattachment site closest to the cross-bridge’s equilibrium positionin the direction towards the Z-line (Fig. 2A).Therefore, followinga short transient phase following muscle shortening or stretching,all history-dependence disappears and muscle force is independentof the contractile history. However, it is generally acknowledged,and uniformly observed, that the steady-state forces followingactive muscle lengthening are increased compared with thecorresponding forces not preceded by stretching. This so-calledresidual force enhancement (Edman et al., 1982) has intriguedmuscle physiologists, as it defies explanation with current cross-bridge thinking, and its molecular origins have eluded satisfactoryexplanation. Below, I will attempt to summarize the observationssurrounding residual force enhancement, identify competingmechanisms, and propose a solution that explains all hithertoobserved observations, but has yet to be proven correct.
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PEVKSoleus N2A
Z-band
Thick filament (myosin)
Z-band
M-li
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Thin filament (actin)
Titin
I-band
A-bandPEVK Fig. 1. Schematic figure of a sarcomere
with the contractile proteins actin (thinfilament) and myosin (thick filament),and the adaptable molecular spring titin,which stabilizes myosin in the centre ofthe sarcomere, provides most of thepassive force in isolated sarcomeresand myofibrils, and changes its stiffnessin an activation (calcium)- and force(cross-bridge attachment)-dependentmanner.
A Equilibrium positionof M-site
Myosin filament
Actin filamentx
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Myosin filament
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Pi releaseInitiatepowerstroke
Hydrolysis
Active sitecleft closure
Transientintermediate
ADPrelease
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Fig. 2. Three classic cross-bridge models. (A) Firstcross-bridge model described by A. Huxley (Huxley,1957) with two states, an attached and a detachedstate. (B) 1971 cross-bridge model with a rotatingcross-bridge head and multiple attachment states(Huxley and Simmons, 1971). (C) Cross-bridge modelproposed by Rayment et al. (Rayment et al., 1993)based on the atomic structure of the cross-bridge headand the actin attachment site. (Adapted and printed withpermission). Pi, inorganic phosphate, i.e. the freephosphate released from ATP when ATP is hydrolysedto ADP.
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Residual force enhancementWhen a muscle, fibre, myofibril or sarcomere is actively lengthened,the steady-state force following lengthening will be greater than thecorresponding force for a purely isometric contraction (Fig. 3). Thisresidual force enhancement has been observed at all structural levelsof muscles (Herzog et al., 2012c), it increases with the magnitudeof stretch (Edman et al., 1982; Herzog et al., 2006), is independentof the speed of stretch (Edman et al., 1982), is greater on thedescending than the ascending limb of the force–length relationship(Morgan et al., 2000; Herzog et al., 2012a), is accompanied by anincrease in passive force (Herzog and Leonard, 2002), and can beeliminated instantaneously when a muscle is deactivated (Abbottand Aubert, 1952). Despite an abundance of consistent observationson force enhancement, and agreement on the properties associatedwith force enhancement, a convincing and generally acceptedmechanism for force enhancement has not been identified. However,three basic ideas have emerged. The first has been associated withthe active force producing cross-bridges, the second with thedevelopment of structural non-uniformities (sarcomere length non-uniformities) when muscles are actively stretched on the descendinglimb of the force–length relationship, and the third with theengagement of passive structural elements.
Cross-bridge mechanism for force enhancementForce enhancement following eccentric muscle action could becaused by an increased force transmission by the cross-bridgesbetween actin and myosin filaments. According to the cross-bridgetheory, there are two ways to increase cross-bridge based forces: (1)by increasing the proportion of attached cross-bridges, or (2) byincreasing the average force per cross-bridge (or a combination ofthe two).
If force enhancement was caused by an increase in the proportionof attached cross-bridges, then residual force enhancement would beassociated with a corresponding increase in muscle stiffness (Fordet al., 1981). However, in the few studies that systematicallyevaluated muscle stiffness in the force-enhanced state, stiffness waseither unchanged from isometric reference contractions or justmarginally increased (Sugi and Tsuchiya, 1988; Rassier and Herzog,2005), suggesting that force enhancement is likely not associatedwith a substantial increase in the proportion of attached cross-bridges. However, findings of increased residual force enhancementin muscle fibres either at cold temperatures or treated with the cross-
bridge inhibitor butanedione monoxime suggest that in thoseexperimental circumstances, force enhancement might be caused inpart by a conversion of weakly to strongly bound cross-bridges(Rassier and Herzog, 2005; Lee and Herzog, 2008a), which wouldbe expected to result in an increase in cross-bridge-based forcewithout a corresponding increase in stiffness.
Force enhancement could also be caused by an increase in theaverage force per cross-bridge while the proportion of attachedcross-bridges remains constant. Because residual force enhancementis long lasting [minutes in myofibrils, where this can be testedwithout the confounding effects of fatigue (Leonard et al., 2010)],this idea is rather unlikely, except if one proposed that cross-bridgesremain attached for minutes following active muscle lengthening, aproposal that cannot be fully rejected at this time, but that seemsrather unlikely. Furthermore, because force enhancement has beenshown to average 285% in single sarcomeres (Leonard et al., 2010),cross-bridges would need to be extended to almost three times oftheir original length to account for such an increase in force. Again,this scenario has not been scientifically eliminated but is highlyunlikely. Therefore, it appears that residual force enhancementcannot be explained easily with cross-bridge action, in accordancewith theoretical considerations (Walcott and Herzog, 2008).
Structural non-uniformity mechanism for force enhancementFor more than three decades, the primary mechanism for forceenhancement was associated with the development of structural non-uniformities, specifically the development of sarcomere length non-uniformities during active muscle lengthening on the descendinglimb of the force–length relationship (Fig. 4). This idea originatedfrom the proposal that muscle segments and sarcomeres werethought to be unstable on the descending limb of the force–lengthrelationship (Hill, 1953; Gordon et al., 1966) because of thepresumed softening behavior of muscle on that part of its workingrange. In the meantime, it has been shown that sarcomeres areperfectly stable on the descending limb of the force–lengthrelationship (Rassier et al., 2003a; Rassier et al., 2003b), and thatthe apparent softening behavior of muscle was an artifact of thestatic, rather than the dynamic, evaluation of the stiffness of muscleson the descending limb of the force–length relationship (Allinger etal., 1996).
The idea of development of sarcomere length non-uniformitiesand their effects on force enhancement were insofar appealing as
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Fig. 3. Force enhancement on three structural levels of skeletal muscle. (A) Force enhancement (FE) in an entire muscle (cat soleus at 37°C), (B) in anisolated myofibril and (C) in a single, mechanically isolated sarcomere. Note also the passive force enhancement (PFE) (A,B). The grey trace in A representsthe isometric reference force and length, while the black traces represent force enhanced states following three stretch magnitudes. The grey trace in B is apassive stretch while the black trace is an active stretch of a myofibril. Finally, in C, the grey trace is the isometric reference force and length, while the blacktrace represents the force-enhanced state following active stretching of a single sarcomere.
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this mechanism could explain force enhancement perfectly withinthe framework of the cross-bridge theory. Furthermore, thesarcomere length non-uniformity theory for force enhancementcould be used to make testable predictions about muscle behaviorfollowing active lengthening. The most important of thesepredictions are that: (1) force enhancement is associated with thedevelopment of sarcomere length non-uniformities while isometricreference contractions are not; (2) force enhancement cannot occuron the ascending limb of the force–length relationship [assarcomeres are stable in that region (Gordon et al., 1966; Hill,1953)]; (3) forces in the enhanced state cannot exceed the maximalisometric forces at the plateau of the force–length relationship; and(4) force enhancement cannot occur in a single sarcomere, as bydefinition, multiple sarcomeres are required for sarcomere lengthnon-uniformities to develop.
All four of these basic predictions of the sarcomere length non-uniformity theory have been rejected by findings in experimentalstudies, suggesting that the development of sarcomere length non-uniformities during active muscle lengthening on the descendinglimb of the force–length relationship is not a major contributor toresidual force enhancement. Specifically, regarding the fourpredictions above, the following observations have been made: (1)forces in the enhanced state have been found to be associated withmore stable (Edman et al., 1982) sarcomere properties and withsimilar or smaller non-uniformities than the corresponding purelyisometric reference contractions (Joumaa et al., 2008a), suggestingthat sarcomere length non-uniformities are present not only in theforce-enhanced but also in the isometric reference states; (2) forceenhancement has been observed on the ascending limb of theforce–length relationships in muscles (Abbott and Aubert, 1952;Morgan et al., 2000) and single fibres (Peterson et al., 2004); (3)forces in the enhanced state can easily exceed the maximal isometricforces at the plateau of the force–length relationship in muscles(Abbott and Aubert, 1952; Schachar et al., 2002), fibres (Lee andHerzog, 2008b), myofibrils (Joumaa et al., 2008a) and singlesarcomeres (Leonard et al., 2010); and (4) force enhancement occursin single sarcomeres and produced forces that exceeded theisometric reference forces by almost a factor of three (Leonard et al.,2010).
In recent years, it has been argued that maybe it is not thesarcomere lengths that matter but the half-sarcomere lengths, andthat the development of half-sarcomere length non-uniformitiesmight produce the observed residual force enhancement observed
consistently in fibre and muscle preparations (Campbell, 2009).However, measurement of half-sarcomere non-uniformities inisolated myofibril preparations suggests that if anything at all, half-sarcomere non-uniformities are smaller in the force-enhancedcompared with the purely isometric reference state (Joumaa et al.,2008a), thus eliminating that possibility. However, in recentexperimental work on half-sarcomere dynamics in isolatedmyofibrils, it was argued that half-sarcomere non-uniformity was amajor contributor to residual force enhancement (Rassier, 2012).Rassier showed maximal half-sarcomere length non-uniformities of12 nm in single sarcomeres and 70 nm in myofibrils in the force-enhanced state. These half-sarcomere length non-uniformities areassociated with an increase in actin–myosin filament overlap (andthus expected increase in force) of ~1.3 and 8.8% for singlesarcomeres and force-enhanced myofibrils, respectively, but theassociated force enhancements were shown to be approximately 20and 50% [see fig. 9 in Rassier (Rassier, 2012)], thus only a tinypercentage of the force enhancement could theoretically beaccounted for by the half-sarcomere length non-uniformities. Moreimportantly, however, although half-sarcomere non-uniformitieswere not systematically evaluated in the isometric referencecontractions, the raw data plots suggest that they were equally, if notmore pronounced in the reference compared with the force-enhanced states, as shown previously by others (Joumaa et al.,2008a), indicating that the half-sarcomere length non-uniformitiesare a regular occurrence of muscle contraction and not a specialfeature of eccentric contraction. Therefore, if sarcomere length non-uniformities were to contribute to force production, one wouldexpect this contribution to be the same for purely isometriccontractions and for isometric contractions following active musclestretching.
In summary, the four major predictions regarding forceenhancement based on the sarcomere length non-uniformity theorycan be rejected based on experimental evidence. Furthermore, forceenhancements of several 100% have been observed in sarcomereand serially arranged half-sarcomere preparations, suggesting thatresidual force enhancement is a true property of sarcomeres andhalf-sarcomeres. Sarcomere and half-sarcomere length non-uniformities appear to be a normal product of any muscularcontraction, rather than a special event that only occurs whenmuscles are actively stretched on the descending limb of theforce–length relationship, thus alternative theories must be found toexplain this evasive property.
Passive mechanism for force enhancementIn 2002, Herzog and Leonard made the observation that forceenhancement in the cat soleus was associated with a passivecomponent that persisted when the muscle was deactivated (Herzogand Leonard, 2002). This observation was termed ‘passive forceenhancement’ (PFE; Fig. 3A,B) and was subsequently observed insingle-fibre and myofibril preparations (Joumaa et al., 2008b; Leeet al., 2007), indicating that this passive component of forceenhancement was a sarcomeric property. We suggested that this PFEmight originate in the structural protein titin (Fig. 1), that it mightcontribute substantially to the total residual force enhancement(Herzog et al., 2012b; Herzog et al., 2012c), and that titin might playa role in muscle contraction that goes beyond its purely passivestructural role. In the next few paragraphs, I will discuss theobservations of titin as the origin of passive and total forceenhancement, and then propose a new model of force regulation thatincludes titin as a ‘contractile’ protein working in tandem with thecontractile proteins actin and myosin.
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Following the discovery of PFE in skeletal muscles (Herzog andLeonard, 2002), we soon focused on the filamentous spring titin asa likely candidate for this property. The reasons for this choice wereinitially purely intuitive. Titin is a long filament that spans half-sarcomeres inserting into the Z-band at one end and the M-line atthe other end (Fig. 1), with known spring-like properties in the I-band region (Kellermayer et al., 1997). Titin is also known toprovide approximately 90–95% of all passive force in singlemyofibrils (Linke, 2000), and a substantial amount of passive forcein skeletal and cardiac muscle (Horowits, 1992). Finally, titin’spassive force depends directly on sarcomere and muscle length(Herzog et al., 2012a), is arranged in parallel with the cross-bridgebased forces, becomes strong when cross-bridge based forcebecomes weak, and provides stability to sarcomeres and myosinfilaments in the centre of sarcomeres (Horowits and Podolsky,1987). Therefore, titin seems uniquely placed to play a role inmuscle force regulation. Even prior to our discovery of PFE, titinhad already been suggested as a candidate for residual forceenhancement (Noble, 1992), as the magnitude of force enhancementincreases with increasing amounts of active muscle lengthening(Edman et al., 1982), it is independent of the speed of lengthening(Edman et al., 1982), and it is partly offset by active shortening ofmuscle immediately preceding stretch (Lee et al., 2001; Rassier andHerzog, 2004), all properties that hint at a passive structural elementthat is engaged upon muscle activation and produces increased forceupon stretch.
Titin is an entropic spring molecule with virtually elasticproperties at short sarcomere lengths (Kellermayer et al., 1997) andat long sarcomere lengths if immunoglobulin (Ig) domain unfoldingin titin is effectively prevented (Granzier et al., 1997; Herzog et al.,2012a; Kellermayer et al., 1997). Titin, like any other springmolecule, can alter its stiffness in essentially two ways: (1) bychanging its inherent stiffness, or (2) by shortening its functionalspring length. The first convincing evidence for a role of titinbeyond its passive force contribution came when it was shown thattitin binds calcium upon muscle activation and that this increasestitin’s stiffness, and thus its force when muscles are stretched (Labeitet al., 2003). This initial result, performed in skinned muscle fibres,was confirmed when calcium activation was shown to increasepassive forces in myofibrils in which cross-bridge based forces wereeliminated through depletion of troponin C from the actin filaments(Joumaa et al., 2008b). Finally, we showed that Ig domain unfoldingin titin requires ~20% more force in the presence of physiologicallyrelevant amounts of calcium (DuVall et al., 2012). All these studiessuggest that upon activation, titin binds calcium at specific locations,thereby increasing its stiffness, and thus its force when stretched.This is a mechanism that could explain residual force enhancementin skeletal muscle contraction (Herzog et al., 2012c). However, theincreases in titin stiffness and force observed in these studies weremerely a fraction of the enhanced forces measured following activemuscle lengthening (Herzog et al., 2006). Therefore, an additionalmechanism explaining the entire magnitude of the residual forceenhancement was needed.
In order to identify the true increase in passive force when amuscle is actively stretched compared with when it is passivelystretched, we elongated single myofibrils from rabbit posas musclesactively (high calcium concentration) and passively (low calciumconcentration) to lengths (6–7 μm per sarcomere) much beyondactin–myosin filament overlap (~3.8 μm per sarcomere) whereactive, actin–myosin-based cross-bridge forces were completelyeliminated, and we found that actively stretched myofibrils hadpassive forces three to four times greater than passively stretched
myofibrils (Fig. 5). Elimination of titin from these preparationsreduced passive forces to a few percent of original and eliminatedany force differences between actively and passively stretchedmyofibrils, indicating that titin was indeed responsible for thesedifferences (Leonard and Herzog, 2010). Stretching the samemyofibrils in a high calcium concentration solution (activestretching) but inhibiting cross-bridge attachments throughbutanedione monoxime reduced the force differences between‘actively’ and ‘passively’ stretched myofibrils to a few percent(result not shown), supporting the idea that calcium binding to titinis just a small part of the enhanced force observed in activelystretched muscle, and that somehow, cross-bridge based forces arerequired to increase the titin-based passive forces when a myofibrilis actively stretched. Finally, when reducing the active forces toapproximately half of maximum, the passive forces upon myofibrilstretching were also reduced to approximately half of those obtainedwhen maximum cross-bridge forces were allowed (Fig. 5).
Combined, these results suggest that titin is a molecular springwhose stiffness, and thus force upon stretch, is increased by calciumbinding upon activation, and that cross-bridge binding and forceproduction increases titin’s stiffness dramatically. It is not clear howthis latter force increase is achieved, but the simplest explanation isthat titin’s proximal regions (those close to the Z-band) attach toactin upon force production, thereby reducing titin’s free springlength, thus increasing its stiffness and force when stretched.Binding of titin to actin at the N2A region or near the PEVK region(Fig. 1) (Herzog et al., 2012b; Herzog et al., 2012c) or winding oftitin onto a rotating thin filament (Monroy et al., 2012; Nishikawaet al., 2012) have been proposed (but by no means proven) as
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Fig. 5. Myofibril force (stress) as a function of sarcomere lengthobtained with slow stretches (0.1 μm s−1 sarcomere−1) of singlemyofibrils beyond actin–myosin filament overlap (lengths greater than3.8 μm sarcomere−1; non-shaded area). Active refers to myofibrils that werestretched while fully activated. Half force refers to experiments in whichmyofibrils were fully activated at ~3.4 μm sarcomere−1 where active forcesare small because of substantial (~70%) loss of actin–myosin filamentoverlap. Passive refers to myofibrils that were stretched passively. Titindepleted refers to passive and active myofibril stretching when titin’s functionwas eliminated by short exposure to trypsin. Note that the actively stretchedmyofibrils have much greater passive forces (beyond actin–myosin filamentoverlap) than the passively stretched myofibrils, indicating a substantialincrease in force in the absence of actin-myosin based cross-bridges. Notefurther that once titin’s function is eliminated, neither passive nor active forceproduction is possible in these myofibril preparations. The shaded areaindicates the region where actin and myosin filaments overlap; the non-shaded area indicates the region of sarcomere lengths where actin–myosinfilament overlap is lost and only passive forces are possible [adapted fromLeonard et al. (Leonard et al., 2010), with permission].
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possible mechanisms for force and stiffness modulation of titin inactively contracting muscles.
In this last section, I will propose a possible model for active andpassive force regulation in skeletal muscle that incorporates thetraditional cross-bridge theory, but adds titin as a third filament withforce-regulating properties to the sarcomere. Although this is merelya proposal for a new mechanism of muscle contraction and forceproduction that needs rigorous testing, it has the advantage that, incontrast to all cross-bridge models, it can predict muscle properties inconcentric, isometric and most importantly in eccentric contractions,and incorporates all findings in the area of residual force enhancementthat have escaped a consistent molecular explanation.
Proposal for a new theory of muscle contraction and forceproductionA revolution in science is described as the replacement of anexisting scientific paradigm with a new and more powerfulparadigm. The new paradigm needs to explain a whole series of thusfar unexplained phenomena while maintaining the successfulpredictions of the old paradigm (Kuhn, 1962). Here, I am proposingan inherently new paradigm of muscle contraction that, in contrastto the cross-bridge theory, not only explains force regulation inisometric and concentric contractions, but also explains forceregulation, stiffness and energetics for eccentric muscle contractions.
Instead of two contractile proteins, actin and myosin, force in thisparadigm is regulated by three sarcomeric proteins, actin, myosin
and titin. While actin and myosin play their usual role and interactvia myosin-based cross-bridges that cyclically attach to specific siteson actin and are driven by ATP hydrolysis (Huxley, 1957), titin actsas a spring that binds calcium upon activation and binds to actinupon cross-bridge attachment. Calcium binding to identified regions,such as the E-rich region in the PEVK domain (Labeit et al., 2003)and selected Ig domains of titin (DuVall et al., 2012), increase titin’sstiffness and thus its force when stretched actively (Leonard andHerzog, 2010b). Selective binding of titin upon cross-bridgeattachment has not been demonstrated directly, but has strongsupport from cross-bridge-dependent increases in titin force(Leonard and Herzog, 2010b) observed in myofibrils stretchedbeyond actin–myosin filament overlap under passive and variousactive force conditions (Leonard and Herzog, 2010b) (Fig. 5).Specifically, the following three-filament theory of muscle forceregulation can explain all experimental observations in isometric andconcentric muscle contractions, and adds a simple explanation tomany phenomena observed in eccentric muscle contraction, such asthe residual force enhancement and the decreased energyrequirement of eccentric contractions.
The three-filament model of muscle contractionIn the three-filament model, activation not only causes cross-bridgesto attach to actin filaments, but also will cause titin to bind calcium atspecific binding sites and bind (at a hitherto unknown site) to actin(Fig. 6). Calcium binding to actin has been identified for at least two
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Fig. 6. Proposed new model of muscle contractionincorporating titin as the third force-regulatingfilament besides actin and myosin. (A) Electronmicrograph of a single myofibril (top panel) with asarcomere isolated (middle panel) and a schematicillustration of the three-filament sarcomere (bottompanel). (B) Schematic proposal of muscle contractionincluding titin as a force-regulating protein. In the toppanel we have two (half) sarcomeres with a short (left)and a long (right) initial length. If passively stretchedfrom these two initial configurations, the initialsarcomere length and passive force is the same(middle panel; passive stretch). If, however, thesarcomeres are activated first at the short and longlengths, respectively (top panel), titin will bind to actinat a more proximal (short initial length) or a moredistal site (long initial length), thus experiencing morestretch for the remnant free spring when the initialsarcomere length is short than when long.Simultaneously, calcium binds to specific sites on titinupon activation, providing an additional increase instiffness to the remnant free spring, thereby addingeven more titin-based force when sarcomeres arestretched actively compared with when they arestretched passively. (C) Active and passive sarcomereforce–length relationships. Note that in this model, thepassive (titin-based) force increases upon activationbecause of the calcium binding to titin and thereduction of titin’s free-spring length. The shift of thepassive force curve upon activation depends cruciallyon the initial sarcomere length, where activationoccurs that will determine where titin binds to actin.FE, force enhancement.
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parts of the protein, the E-rich portion of the PEVK region (Labeit etal., 2003), and specific immunoglobulin domains (DuVall et al.,2013), while binding of titin to actin upon force production has notbeen experimentally verified in situ. However, because we know thattitin’s contribution to active force requires cross-bridge binding andactin–myosin-based force [rather than just calcium activation(Leonard and Herzog, 2010b)], a possible explanation might includea mechanism whereby the regulatory proteins troponin andtropomyosin free up titin binding sites on actin upon strong cross-bridge binding, thus allowing titin’s free-spring length to shorten inthe presence of cross-bridge forces but not in the presence ofactivation (calcium) alone. For eccentric contractions, the increase instiffness in titin will result in an increased force upon stretch, whichcomes at virtually no additional energy cost. The actual forcetransmitted by titin will depend primarily on the binding site of titinto actin. The more distal this titin binding site, the shorter the free-spring length, and the greater titin’s stiffness and force upon stretch.
Fig. 6 illustrates this scenario. In the top row, two half-sarcomereswith an actin, myosin and titin filament are shown for a shortpassive (left) and a long passive (right) half-sarcomere. If passivelystretched (no titin binding to actin) to a certain length (middle row),the elongation of titin for both initial sarcomere lengths is the same,and the force transmitted by titin is the same as well. However, ifthe two half-sarcomeres are stretched actively (bottom row), titinwill have attached at a more proximal (closer to the Z-line) locationto actin for the initially short half-sarcomere compared with theinitially long half-sarcomere. Therefore, if both half-sarcomeres arestretched actively to the same length, the stretch of the remnant free-spring titin segments is greater for the initially short than the initiallylong half-sarcomere, and thus will produce a greater titin force.However, if titin indeed binds to actin in a force-dependent manner,where this binding occurs, and whether this binding can occur atmultiple locations or occurs in an altogether different way(Nishikawa et al., 2012; Herzog et al., 2012b; Herzog et al., 2012c),needs careful study, but will likely be known within the next 3 yearsthrough ongoing titin segment labeling work in our laboratory andin the laboratory of Dr Nishikawa at Northern Arizona University.
If the proposal of titin’s force regulation in a calcium- and force-dependent manner is confirmed, it would explain severalunaccounted properties of skeletal muscle contraction. For example,the following observations would have a ready explanation: (1) theresidual force enhancement observed following active musclestretching; (2) the energetic efficiency of eccentric musclecontraction; (3) the stability of sarcomeres and muscle segments onthe descending limb of the force–length relationship; and (4) thestability of myosin in the centre of the sarcomere.
However, aside from these properties, the proposed contributionof titin to active force production would also have some intriguingimplications for active and passive force regulation in skeletalmuscles and associated properties. The primary examples arethreefold. First, it would allow for passive muscle elongation againstlittle resistance, while providing great passive (titin-based) resistanceat virtually no energy cost in eccentric contractions. Titin’s activerole, therefore, might be similar to that observed in the catchmechanism of molluscan muscle by the titin-like protein twitchin,which connects myosin with actin and establishes a long-lastingforce state (Butler et al., 2001) at essentially no energy cost. Second,it would protect muscles against muscle injuries on the descendinglimb of the force–length relationship. Third, it would provideincreases in force when the actin–myosin-based cross-bridge forcesdecrease, thereby allowing for a greater working range ofsarcomeres with useful force contributions.
In summary, the cross-bridge theory has been the paradigm ofchoice for muscle contraction mechanisms for the past half century(Huxley, 1957). It relies on force regulation through the contractileproteins actin and myosin. However, it does not account for manyproperties of muscles observed during eccentric contractions. Bystudying one of these unexplained properties, residual forceenhancement, we stumbled across the force-regulating role of titin,and although titin’s role is by no means understood in detail, it isnow accepted that titin can change its stiffness in an activation(calcium)-dependent manner. The molecular details of this forceregulation will need further elucidation, and thus the modelproposed here for contraction mechanisms and force regulation inskeletal muscles (Fig. 6) needs to be seen as such – a proposal ratherthan a fully established theory. However, the beauty of this proposalis that it incorporates and asserts the cross-bridge theory, and merelyadds a third myofilament, titin, to the contractile mechanism.
A great advantage of this three-filament model is that it endowsmuscle with some elegant properties: an increase in force for onesystem (titin) when force in the other system (actin-myosin)decreases at long muscle lengths, elongation of passive muscleagainst little resistance and elongation of active muscle against great(passive) resistance that comes at no extra (energetic) cost, and anexplanation for the residual force enhancement, a property that hasbeen identified prior to the formulation of the cross-bridge andsliding filament theories (Abbott and Aubert, 1952), but has eludedconvincing explanation for decades.
Competing interestsThe author declares no competing financial interests.
FundingW.H. is funded by the Canadian Institutes of Health Research, the NaturalSciences and Engineering Research Council of Canada, The Killam Foundationand The Canada Research Chair Programme.
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